Presence of Functionally Active Cytolethal Distending Toxin Genes on a Conjugative Plasmid in a Clinical Isolate of Providencia rustigianii.

Providencia rustigianii is potentially enteropathogenic in humans. Recently, we identified a P. rustigianii strain carrying a part of the cdtB gene homologous to that of Providencia alcalifacines that produces an exotoxin called cytolethal distending toxin (CDT), encoded by three subunit genes (cdtA, cdtB, and cdtC). In this study, we analyzed the P. rustigianii strain for possible presence of the entire cdt gene cluster and its organization, location, and mobility, as well as expression of the toxin as a putative virulence factor of P. rustigianii. Nucleotide sequence analysis revealed the presence of the three cdt subunit genes in tandem, and over 94% homology to the corresponding genes carried by P. alcalifaciens both at nucleotide and amino acid sequence levels. The P. rustigianii strain produced biologically active CDT, which caused distension of eukaryotic cell lines with characteristic tropism of CHO and Caco-2 cells but not of Vero cells. S1-nuclease digested pulsed-field gel electrophoresis followed by Southern hybridization analysis demonstrated that the cdt genes in both P. rustigianii and P. alcalifaciens strains are located on large plasmids (140 to 170 kb). Subsequently, conjugation assays using a genetically marked derivative of the P. rustigianii strain showed that the plasmid carrying cdt genes in the P. rustigianii was transferable to cdt gene-negative recipient strains of P. rustigianii, Providencia rettgeri, and Escherichia coli. Our results demonstrated the presence of cdt genes in P. rustigianii for the first time, and further showed that the genes are located on a transferable plasmid, which can potentially spread to other bacterial species.

Critical role of the RpoE stress response pathway in polymyxin resistance of Escherichia coli.

OBJECTIVES: Polymyxins, including colistin, are the drugs of last resort to treat MDR bacterial infections in humans. In-depth understanding of the molecular basis and regulation of polymyxin resistance would provide new therapeutic opportunities to combat increasing polymyxin resistance. Here we aimed to identify novel targets that are crucial for polymyxin resistance using Escherichia coli BL21(DE3), a unique colistin-resistant model strain. METHODS: BL21(DE3) was subjected to random transposon mutagenesis for screening colistin-susceptible mutants. The insertion sites of desired mutants were mapped; the key genes of interest were also inactivated in different strains to examine functional conservation. Specific genes in the known PmrAB and PhoPQ regulatory network were inactivated to examine crosstalk among different pathways. Lipid A species and membrane phospholipids were analysed by normal phase LC/MS. RESULTS: Among eight mutants with increased susceptibility to colistin, five mutants contained different mutations in three genes (rseP, degS and surA) that belong to the RpoE stress response pathway. Inactivation of rpoE, pmrB, eptA or pmrD led to significantly increased susceptibility to colistin; however, inactivation of phoQ or eptB did not change colistin MIC. RpoE mutation in different E. coli and Salmonella resistant strains all led to significant reduction in colistin MIC (16-32-fold). Inactivation of rpoE did not change the lipid A profile but significantly altered the phospholipid profile. CONCLUSIONS: Inactivation of the important members of the RpoE regulon in polymyxin-resistant strains led to a drastic reduction in polymyxin MIC and an increase of lysophospholipids with no change in lipid A modifications.

Cefixime-tellurite-deoxycholate tryptic soy broth (CTD-TSB), a selective enrichment medium, for enhancing isolation of Escherichia albertii from wild raccoon fecal samples.

AIM: The aim of this study was to develop a selective enrichment broth for efficient isolation of Escherichia albertii. METHODS AND RESULTS: A total of 412 raccoon rectal swabs suspended in PBS (phosphate-buffered saline) were tested by a real-time PCR to quantify the number of E. albertii followed by its isolation. The number of E. albertii in the PBS suspension strongly affected the isolation rate (1.2%-89%), which notably dropped (≤33%) when the number was <4 log10 CFU ml-1. However, enrichment of PBS suspension containing raccoon feces in tryptic soy broth containing cefixime, tellurite, and deoxycholate (CTD-TSB), the selective medium developed in this study, remarkably improved the isolation efficiency (up to 48%) of E. albertii. CONCLUSIONS: CTD-TSB is a useful enrichment culture medium for E. albertii and contributes to increase its isolation rate.

Prevalence, Serovar, and Antimicrobial Resistance of Nontyphoidal Salmonella in Vegetable, Fruit, and Water Samples in Ho Chi Minh City, Vietnam.

In this study, we investigated the prevalence, serovar distribution, and antimicrobial resistance pattern of Salmonella isolates from vegetable, fruit, and water samples in Ho Chi Minh City, Vietnam. Salmonella was detected in 75% (30/40), 57.1% (12/21), 17.5% (28/160), and 2.5% (1/40) of river water, irrigation water, vegetable, and ice water samples, respectively. However, no Salmonella was isolated from 160 fruit and 40 tap water samples examined. A total of 102 isolates obtained from 71 samples belonged to 34 different serovars, of which Salmonella Rissen was the most prevalent, followed by Salmonella London, Salmonella Hvittingfoss, and Salmonella Weltevreden. Certain Salmonella serovars such as Newport, Rissen, and Weltevreden were isolated from both vegetable and water samples. Antimicrobial resistance was most commonly observed against tetracycline (35.3%), followed by chloramphenicol (34.3%), ampicillin (31.4%), trimethoprim/sulfamethoxazole (23.5%), and nalidixic acid (10.8%). Of 102 isolates analyzed, 52 (51%) showed resistance to at least 1 antimicrobial class whereas 27 (26.5%) showed multidrug resistant (MDR) phenotype, being resistant to at least three different classes of antimicrobials. Determination of the presence and type of ß-lactamase genes showed the cooccurrence of blaTEM-1 and blaCMY-2 in one Salmonella Agona isolate from a river water sample. Taken together, these data indicated that both environmental water and vegetables were contaminated with Salmonella, including MDR strains, and that environmental water used in irrigation might have been the source of Salmonella contamination in the vegetables.

Prevalence of Escherichia albertii in Raccoons (Procyon lotor), Japan.

Natural reservoirs of Escherichia albertii remain unclear. In this study, we detected E. albertii by PCR in 248 (57.7%) of 430 raccoons from Osaka, Japan, and isolated 143 E. albertii strains from the 62 PCR-positive samples. These data indicate that raccoons could be a natural reservoir of E. albertii in Japan.

Development of a bivalent food poisoning vaccine: augmented antigenicity of the C-terminus of Clostridium perfringens enterotoxin by fusion with the B subunit of Escherichia coli Shiga toxin 2.

Food poisonings caused by Clostridium perfringens and Shiga toxin (Stx)-producing Escherichia coli (STEC) occur frequently worldwide; however, no vaccine is currently available. Therefore, we aimed to develop a bivalent vaccine against C. perfringens and STEC infections. Although it has been considered that the C-terminal region of C. perfringens enterotoxin (C-CPE) could be a good vaccine antigen to block the binding to its receptor, it was insufficient for induction of a protective immune response because of the low antigenicity. However, the fusion of C-CPE with Stx2 B subunit (Stx2B) augmented the antigenicity of C-CPE without affecting the antigenicity of Stx2B. Indeed, high levels of C-CPE-specific neutralizing IgG were found in the serum of mice immunized with the fusion protein Stx2B-C-CPE. Additionally, comparable and substantial levels of Stx2B-specific neutralizing IgG were induced in mice receiving Stx2B-C-CPE or Stx2B alone. These antibody responses against C-CPE and Stx2B lasted for at least 48 weeks, which were sufficient for protective immunity in vitro and in vivo, indicating that Stx2B-C-CPE could induce long-term protective immunity. As an underlying mechanism, ex vivo stimulation with Stx2B, but not with C-CPE, induced cytokine production from splenic T cells collected from mice immunized with Stx2B-C-CPE, suggesting that Stx2B-specific, but not C-CPE-specific, T cells were induced by the immunization with Stx2B-C-CPE and plausibly promoted immunoglobulin class switching of both Stx2B- and C-CPE-specific B cells from IgM to IgG. These findings collectively indicate that Stx2B-C-CPE is a T-cell-antigen-supplement-type bivalent vaccine, which could be an efficient against C. perfringens and STEC infections.

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3' end of the tRNA genes pheV or pheR The length of the target site was 52 or 55 bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.

Phenotypic and molecular characterization of Escherichia albertii: Further surrogates to avoid potential laboratory misidentification.

Escherichia albertii is an emerging gastrointestinal pathogen, related to Escherichia coli, which can be misidentified as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), due to the presence of the eae gene in E. albertii. The aim of this study was to verify our hypothesis that E. coli cytolethal distending toxin-II (Eccdt-II) gene-positive E. coli is E. albertii and to accumulate the data regarding the bacteriological characteristics of E. albertii. For these purposes, we attempted to detect E. albertii in eae gene-positive bacteria previously identified as E. coli and to examine if re-identified E. albertii contained Eccdt-II-homologous gene and remaining eae gene-positive E. coli did not. A total of 373 eae gene-positive E. coli strains were analyzed by biochemical tests, multilocus sequence analysis and an E. albertii-specific PCR. The strains re-identified as E. albertii were also examined for the presence of cdt genes by using 32P-labled DNA probes, followed by their toxin-typing. Of the 373 strains, 17 were re-identified as E. albertii by three above-mentioned methods. Furthermore, all the 17 re-identified E. albertii possessed cdt genes highly homologous to Eccdt-II and Eacdt genes. Moreover, Eccdt-I or both Eccdt-I and stx2f genes were detected in two re-identified E. albertii strains. However, the remaining 356 strains did not carry such cdt genes. These data indicate that all re-identified E. albertii isolates specifically carried cdt genes homologous to Eccdt-II and Eacdt genes. We suggest that Eccdt-II gene-positive E. coli may be identical to E. albertii.

Novel Cholera Toxin Variant and ToxT Regulon in Environmental Vibrio mimicus Isolates: Potential Resources for the Evolution of Vibrio cholerae Hybrid Strains.

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in V. mimicusctx-positive (ctx+) strains (i.e., V. mimicus strains containing ctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx+V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains.IMPORTANCE Natural diversification of CTXФ and ctxAB genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of V. cholerae This report, showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnv and of a CTXФET in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

Phylogenetic Characterization of Salmonella enterica Serovar Typhimurium and Its Monophasic Variant Isolated from Food Animals in Japan Revealed Replacement of Major Epidemic Clones in the Last 4 Decades.

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) and its monophasic variant (Salmonella 4,[5],12:i:-) are the major causes of gastroenteritis in both humans and animals. Pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis have been used widely as subtyping methods for these pathogens in molecular epidemiological analyses, but the results do not precisely reflect phylogenetic information. In this study, we performed a phylogenetic analysis of these serovars using whole-genome sequencing data and identified nine distinct genotypic clades. Then, we established an allele-specific PCR-based genotyping method detecting a clade-specific single nucleotide polymorphism to rapidly identify the clade of each isolate. Among a total of 815 isolates obtained from cattle in Japan between 1977 and 2017, clades 1, 7, and 9 contained 77% of isolates. Obvious replacement of the dominant clone was observed five times in this period, and clade 9, which mostly contains Salmonella 4,[5],12:i:-, is currently dominant. Among 140 isolates obtained from swine in Japan between 1976 and 2017, clades 3 and 9 contained 64% of isolates. Clade 9 is the latest clone as is the case in cattle isolates. Clade 9 is similar to an epidemic clone from Europe, which is characterized by sequence type 34 (ST34), chromosomal Salmonella genomic island 3, and a composite transposon containing antimicrobial resistance genes. The increased prevalence of clade 9 among food animals in Japan might be a part of the pandemic of the European Salmonella 4,[5],12:i:- clone.

Association of cytolethal distending toxin-II gene-positive Escherichia coli with Escherichia albertii, an emerging enteropathogen.

Cytolethal distending toxin (CDT)-producing Escherichia coli have been isolated from patients with diarrhea, sepsis and urinary tract infection. CDT of E. coli is divided into five types (CDT-I through CDT-V) based on differences in amino acid sequences and its genomic location. However, in our recent studies, a few strains of cdt-II gene-positive bacteria, initially identified as atypical E. coli, were re-identified as Escherichia albertii, an emerging enteropathogen, by extensive characterization including multilocus sequence (MLS) analysis and sugar utilization tests. This finding prompted us to investigate if bacteria previously identified as cdt-II gene-positive E. coli might be E. albertii. In the present study, we therefore re-examined the identity of 20 cdt-II gene-positive bacteria isolated from children with diarrhea, which were initially identified as atypical E. coli. By extensive sugar utilization tests, these bacteria showed a closer relatedness to E. albertii than E. coli, because they did not ferment any of the tested sugars including dulcitol, lactose, d-melibiose, l-rhamnose and d-xylose. Further, both phylogenetic analyses based on nucleotide sequences of 7 housekeeping genes (MLS analysis) and rpoB gene showed that all the cdt-II gene-positive bacteria belonged to a distinct lineage of E. albertii from those of E. coli and Shigella boydii. They were also positive by an E. albertii-specific PCR. Taken together, these data suggest that cdt-II gene-positive bacteria previously identified as E. coli are actually E. albertii. Therefore, we suggest a new definition for cdt-II gene-positive E. coli as E. albertii with the inclusion of CDT-II in E. albertii CDT.

Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin.

Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals.

Molecular characterization of cytolethal distending toxin gene-positive Escherichia coli from healthy cattle and swine in Nara, Japan.

BACKGROUND: Cytolethal distending toxin (CDT)-producing Escherichia coli (CTEC) has been isolated from patients with gastrointestinal or urinary tract infection, and sepsis. However, the source of human infection remains unknown. In this study, we attempted to detect and isolate CTEC strains from fecal specimens of healthy farm animals and characterized them phenotypically and genotypically. RESULTS: By PCR analysis, the cdtB gene was detected in 90 and 14 out of 102 and 45 stool specimens of healthy cattle and swine, respectively, and none from 45 chicken samples. Subtypes of the cdtB genes (I to V) were further examined by restriction fragment length polymorphism analysis of the amplicons and by type-specific PCRs for the cdt-III and cdt-V genes. Of the 90 cdtB gene-positive cattle samples, 2 cdt-I, 25 cdt-III, 1 cdt-IV, 52 cdt-V and 1 both cdt-III and cdt-V gene-positive strains were isolated while 1 cdt-II and 6 cdt-V gene-positive were isolated from 14 cdtB positive swine samples. Serotypes of some isolates were identical to those of human isolates. Interestingly, a cdt-II gene-positive strain isolated from swine was for the first time identified as Escherichia albertii. Phylogenetic analysis grouped 87 E. coli strains into 77 phylogroup B1, 6 B2, and 4 D, respectively. Most of the B1 strains harbored both lpfAO113 and ehaA. Three and twenty-two cdt-V gene-positive strains harbored eaeA and stx genes, respectively, and seven possessed cdt-V, stx and subAB genes. The cnf2 gene, normally present in cdt-III gene-positive strains, was also detected in cdt-V gene-positive strains. CONCLUSIONS: Our results suggest that healthy cattle and swine could be the reservoir of CTEC, and they could be a potential source of human infections.

Seasonality of detection rate of Escherichia albertii in wild raccoons (Procyon lotor) in Osaka, Japan.

Escherichia albertii has increasingly been recognized as an important emerging zoonotic enteropathogen. Raccoon is shown to be one of the most vital reservoirs of this pathogen. E. albertii has been detected in 993 (62%) out of 1,606 wild raccoons in Osaka, Japan from 2017 to 2020 by Eacdt-PCR. The detection rate of E. albertii was increased from May to December (winter) and gradually decreased from January to April (spring). Furthermore, we could isolate E. albertii from 30% (196/664) of Eacdt-PCR positive samples and the monthly isolation rate seems to correlate with its detection rate. These data indicate that there is a seasonality regarding the prevalence of E. albertii in wild raccoon being higher in winter and lower in spring.

Improved molecular identification of Strongyloides myopotami in nutrias using fecal samples.

Strongyloides myopotami is an intestinal nematode parasite of nutrias. Identification of S. myopotami is conducted based on the morphological characteristics of adult worms or cultured larvae. To widely and effectively understand the infection in nutrias, it would be preferable to develop the molecular identification using a few grams of the feces. Here, we attempted to identify S. myopotami using DNA extracted from eggs obtained from fecal samples. Among previously reported primer pairs targeting the 18S rRNA gene of Strongyloides spp., most could not be successful. We newly designed primers that successfully amplified the partial sequences in S. myopotami, resulting in being sequenced. Our simple protocol can be useful in nationwide surveys for clarifying the risk of human infection.

Detection of prolong excretion of Escherichia albertii in stool specimens of a 7-year-old child by a newly developed Eacdt gene-based quantitative real-time PCR method and molecular characterization of the isolates.

Escherichia albertii is an emerging zoonotic foodborne pathogen. The clinical significance of this bacterium has increasingly been recognized worldwide. However, diagnostic method has not yet been established and its clinical manifestations are not fully understood. Here, we show that an Eacdt gene-based quantitative real-time PCR (qRT-PCR) developed in this study is 100% specific and sensitive when tested with 39 E. albertii and 36 non-E. albertii strains, respectively. Detection limit of the real-time PCR was 10 colony forming unit (CFU) and 1 pg of genomic DNA per PCR tube. When E. albertii was spiked with 4 × 100-106 CFU per mL to stool of healthy person, detection limit was 4.0 × 103 and 4.0 CFU per mL before and after enrichment culture, respectively. Moreover, the qRT-PCR was able to detect E. albertii in five children out of 246 (2%) but none from 142 adults suffering from gastroenteritis. All five E. albertii strains isolated carried eae and paa genes, however, only one strain harbored stx2f genes. Long-term shedding of stx2f gene-positive E. albertii in a child stool could be detected because of the qRT-PCR developed in this study which might have been missed if only conventional PCR and culture methods were employed. Furthermore, E. albertii isolated from siblings with diarrhea showed clonality by PFGE analysis. Taken together, these data suggest that the Eacdt gene-based qRT-PCR developed for the detection of E. albertii is useful and will assist in determining the real burden and clinical manifestation of E. albertii infections.

QcrC is a potential target for antibody therapy and vaccination to control Campylobacter jejuni infection by suppressing its energy metabolism.

Introduction: Campylobacter spp. are a public health concern, yet there is still no effective vaccine or medicine available. Methods: Here, we developed a Campylobacter jejuni-specific antibody and found that it targeted a menaquinol cytochrome c reductase complex QcrC. Results: The antibody was specifically reactive to multiple C. jejuni strains including clinical isolates from patients with acute enteritis and was found to inhibit the energy metabolism and growth of C. jejuni. Different culture conditions produced different expression levels of QcrC in C. jejuni, and these levels were closely related not only to the energy metabolism of C. jejuni but also its pathogenicity. Furthermore, immunization of mice with recombinant QcrC induced protective immunity against C. jejuni infection. Discussion: Taken together, our present findings highlight a possible antibody- or vaccination-based strategy to prevent or control Campylobacter infection by targeting the QcrC-mediated metabolic pathway.

Novel cholix toxin variants, ADP-ribosylating toxins in Vibrio cholerae non-O1/non-O139 strains, and their pathogenicity.

Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.

Endotoxin-Free Stx2B-C-CPE Vaccine and Its Optimized Adjuvant Regimen for Preventing Food Poisoning.

BACKGROUND: Clostridium perfringens and Shiga toxin (Stx)-producing Escherichia coli (STEC) are common causes of food poisoning. We previously demonstrated the efficacy of Stx2B-C-CPE, a fusion protein of the C-terminal region of C. perfringens enterotoxin (C-CPE) and Shiga toxin 2 B subunit (Stx2B), as a bivalent vaccine against C. perfringens and STEC infections. METHODS: Here, we applied an E. coli expression system and Triton X-114 phase separation to prepare tag- and endotoxin-free Stx2B-C-CPE for use in vaccine formulations. RESULTS: As we anticipated, endotoxin removal from the purified antigen reduced both Stx2B- and C-CPE-specific IgG antibody responses in subcutaneously immunized mice, suggesting that endotoxin contamination influences the immunological assessment of Stx2B-C-CPE. However, the combined use of aluminum and Alcaligenes lipid A adjuvants improved IgG antibody responses to the injected antigen, thus indicating the suitability of purified Stx2B-C-CPE for vaccine formulation. CONCLUSIONS: Our current findings provide important knowledge regarding the design of an effective commercial Stx2B-C-CPE vaccine.

Molecular characterizations of cytolethal distending toxin produced by Providencia alcalifaciens strains isolated from patients with diarrhea.

Cytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides--CdtA, CdtB, and CdtC--encoded by the genes cdtA, cdtB, and cdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles of cdtB (Cdt-I through Cdt-V) in Escherichia coli and used the assay to investigate the prevalence and characteristic of CDT-producing E. coli in children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206-215, 2009). In these assays, two untypable cdtB genes were detected and the organisms harboring the cdtB gene were identified as Providencia alcalifaciens (strains AH-31 and AS-1). Nucleotide sequence analysis of the cdt gene cluster revealed that the cdtA, cdtB, and cdtC genes of P. alcalifaciens are of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of the cdt genes among closely related species, the presence of cdt genes was screened in various Providencia spp. by colony hybridization assay, and the cdt gene cluster was found in only limited strains of P. alcalifaciens. Genome walking revealed that the cdt gene cluster of P. alcalifaciens is located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT of P. alcalifaciens (PaCDT) showed some homology with the CDT of Shigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells, E. coli CDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G(2)/M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinical P. alcalifaciens strains could produce variants of the CDTs compared.